DESCRIPTION: (Verbatim from the Applicant's Abstract) Dopamine neurons in the substantia nigra die prematurely in Parkinson's disease, and apoptotic death has been detected postmortem. The cause of apoptosis is unknown. Two prominent, yet unsubstantiated hypotheses are that apoptosis is mediated by oxidative stress or inappropriate reentry into the cell cycle; both conditions may be initiated by insufficient neurotrophic support from striatal target cells. We will address this knowledge gap by studying mechanisms underlying apoptosis in cultured catecholamine cells, where conditions can me more tightly controlled than in animals. Cells to be studied include pheochromocytoma (PC12), sympathetic neurons, and fetal nigral neurons. These models have in vivo relevance, since apoptotic death of nigral dopamine neurons during development may be due to insufficient trophic support from striatal target cells; this mechanism has been suggested as a causal cell death factor in Parkinson's disease. Tetrahydrobiopterin (BH4) is an essential regulatory cofactor for tyrosine hydroxylase and nitric oxide (NO) synthase in the synthesis of catecholamines and NO, which will not occur if BH4 is lacking. Catecholamine cells contain among the highest concentrations of BH4. The metabolism of BH4, catecholamines, and NO can generate damaging reactive oxygen species (ROS). Our preliminary data shows that apoptotic death of differentiated neuron-like PC12 cells during nerve growth factor withdrawal is directly proportional to the intracellular level of BH4. Thus, we hypothesize that the endogenous level of BH4 supports catecholamine cell functions under normal conditions and promotes apoptotic death when trophic support is withdrawn. Our working hypothesis and specific aim is: Intracellular BH4 mediates apoptosis and death of catecholamine cells deprived of trophic support. While the complex processes of apoptosis occur in many cell types, we will focus on catecholamine cells and the direct and indirect interactions of BH4 with pivotal mediators of apoptosis of these cells during insufficient trophic support. These mediators include oxidative stress, oncogenes that can initiate fatal reentry into the cell cycle, and critical cysteine proteases (caspases) mediating apoptosis. Our studies will identify the mechanism of BH4 involvement in apoptosis by testing for: 1) elevation of ROS and the source (BH4, catecholamines, or NO); 2) altered expression of the apoptotic-mediating oncogenes, pS3, c-myc, and the bc1-2 family (bax, bak, bc1-2, bcl-xl), and 3) activation of caspases 2,3, and 9. Apoptosis will be monitored by: a) number of living and dead cells; and b) fluorescent quantitation of cells at different stages of apoptosis. These studies may will reveal novel apoptotic regulatory mechanisms and provide new avenues for therapeutic approaches to promote neuronal survival and prevent debilitating neurological diseases, such as Parkinson's disease.